1. Field of the Invention
The present invention relates to a method of activating of an enzyme such as lipase, etc., to reaction of such activated enzyme including reformation of fat, and further to a method of deactivating the enzyme once it is activated.
2. Description of Related Art
Conventionally, the production of many useful products has been achieved with use of enzymes. In particular, lipase was widely used for reforming fat so as to produce edible oil, soap, glycerin, dairy or milk products, etc., because of the versatility thereof.
Today where resources, energy, and ecological problems are discussed daily on a global scale, many expectations are applied to efficient and safe production of important materials (i.e., fatty acids, etc.) in the chemical industry, as well as to high-performance materials to which much attention is paid in the fields of pharmaceuticals and foodstuffs, with the use of the enzyme which can be represented by lipase.
As a method for obtaining a target material with use of the enzyme, there can be listed a hydrolysis reaction with use of lipase, transesterification, and ester synthesis or composing reaction, etc.
As a different method for reforming those fats, conventionally, an organic synthetic method using high temperature is mainly used or practiced. However, such method cannot be applied to materials which are unstable at high temperatures, and it is impossible to reproduce the target material with high efficiency due to poor enatioselectivity. Further, with the enzyme method but not with the organic synthetic method, since a residual activating material which may have toxicity or may cause a side reaction is used for increasing the activity of the enzyme, a problem arises in particular in the field of foodstuffs.
Therefore, production of the useful materials by catalytic reaction with use of an enzyme with high efficiency and with safety is considered.
For example, in a case of the catalytic reaction with use of lipase, it was reported that it proceeds with the useful enatioselectivity where 1-phenylethylelaurate (R-soma) is synthesized on a basis of 1-phenylethanol and lauric acid, as the ester synthesis between fatty acid and asymmetric alcohol.
Also, aside from the enatioselectivity, if the lipase having selectivity in a reacting portion, in particular participating in ester bonding between a first order (1st order) and a third order (3rd order) of triglyceride is used, it is possible to put the target fatty acid into the positions of only the first and the third orders. However, in a case of a chemical method using an inorganic catalyst, there is no such selectivity, therefore the fatty acid is put in at random.
However, most enzymes, such as lipase, show only a hydrolysis reaction in water solution, and will not proceed to the reaction if a powder thereof is simply dispersed into an organic solvent by itself.
Then, for conducting the transesterification and the ester composing reaction, which are industrially valuable, with high efficiency in a non-aqueous or nano-aqueous system or condition, there are proposed various methods, including a method in which the enzyme is fixed onto a carrier, a method in which the surface the enzyme is made hydrophobic with a covalent bond, and a method which the enzyme is modified with a surfactant or fatty acid.
For fixing the enzyme onto the carrier, there are already known a method in which the enzyme is physically absorbed onto the surface of an inorganic carrier, such as alumina particles, and a method in which the enzyme is fixed with the covalent bond onto the surface of latex particles or silica particles.
For making the surface of the enzyme hydrophobic, there is already known a method in which polyethylene glycol is bonded on the surface of the enzyme with the covalent bond, and there is also known a method in which the enzyme is made soluble into an organic solvent.
Further, for modifying the lipase (i.e., the enzyme) with the fatty acid or the surfactant, there is known a method in which the enzyme is bonded with the fatty acid or the surfactant through an interaction which seems to be electrostatic, hydrophilic or hydrophobic.
With the method of fixing the enzyme onto the carrier, however, it is impossible to conduct the transesterification or the ester composing reaction with such high efficiency. Further, there results a solid enzyme which cannot be applied to the production of foodstuffs.
With the method of making the surface of an enzyme hydrophobic, however, the enzyme is easily deactivated during the process of making it hydrophobic, and the activity which can be actually obtained therefrom is low. The chemical(s) used for making the surface of the enzyme hydrophobic cannot be used for producing the foodstuffs, thereby restricting the utilization of the enzyme itself.
With the method of modifying the enzyme with the fatty acid and/or the surfactant, the enzyme shows interesterification activity under the condition where it forms a compound with the fatty acid and/or the surfactant. However the enzyme itself does not show the activity. In other words, since it is impossible to remove the modification(s) of the fatty acid and/or the surfactant from a reaction system, there is a probability that the fatty acid and/or the surfactant may participate in some kind reaction so that the modification is mixed or added into a product or a secondary product.
It is considered that lipase has an active site and a xe2x80x9clidxe2x80x9d covering the active site, and it is low in interesterification activity under the condition where the lid is closed as shown in FIG. 1(a), while high in interesterification activity under the condition where the lid is open as shown in FIG. 1(b).
The lipase is considered to close the lid thereof in water solution so that it is in the condition where a substrate (i.e., water) cannot enter into the active site. However, in a water solution mixed with fat, it is considered that the lipase gathering on a boundary surface between water and fat opens the lid thereof so as to accelerate the hydrolysis reaction. By the way, the above consideration can be confirmed by the fact that hydrolysis is increased rapidly by a fat concentration exceeding a level of saturation of fat.
As mentioned previously, the reaction will not occur even if the powder of lipase is dispersed into the organic solvent. However, the lipase being modified with the fatty acid shows the activity of the enzyme in the organic solvent.
This is inferred by the inventors, as shown in FIG. 2, because the hydrophobic portion of the lipase enters into double layers of fat being approximately 5 nm in the thickness thereof, while the hydrophilic portion thereof forms a compound protruding from double layers of fat.
According to the present invention, upon the above inference, it is considered that the enzyme should show the activity thereof if it is kept with the lid open in the non-aqueous or nano-aqueous system, where no boundary surface exists with a water phase, thereby accomplishing the present invention.
Namely, according to the present invention, there is provided a method for producing activated enzyme, comprising the following steps:
adding enzyme to water and fat phases of a multi-phase system;
activating the enzyme due to a function of a fat-and-water boundary surface between the water and fat phases; and
removing the water and fat phases while maintaining an activated condition of the enzyme.
As a concrete method for adding the enzyme into the two-phase system of the water phase and the fat phase, there can be considered a method of adding the fat phase to a water solution into which the enzyme has been dissolved in advance.
And, as a means for removing the fat phase and the water phase while keeping the activating condition of the enzyme, freeze-drying (or lyophilization) is appropriate.
Further, a concrete example of the enzyme can be listed as lipase, and as the fat phase can be listed a volatile one, such as tetradecane.
Further, when activating, the pH of the water phase (buffer solution) is preferably maintained in a vicinity of neutrality, and the addition amount of said tetradecane is to be from 1% to 10% of the volume of the buffer solution.
By contacting the activated enzyme obtained from the method according to the present invention described above with fat in non-aqueous system or nano-aqueous system, reforming can be conducted, such as transesterification reaction, ester composing reaction, etc.
Further, in a method for inactivating an activated enzyme, according to the present invention, the activated enzyme, which is activated by means of the above method or the other method(s), is dispersed and stirred into a buffer solution in which there exists no boundary surface between fat and water.